Expression of Deer Adenovirus Spike Protein By Dang

Expression of Deer Adenovirus Spike Protein By: Dang Duong

Background Introduction • Adenoviruses infect wide variety of animals – Wild & Domestic – Pathogenic in Deer – Symptoms of AV are ulcers and abscesses in the mouth and throat – Acute Symptoms would be rapid breathing, diarrhea, foaming at the mouth – Death can occur with 3 -5 days from the time of the exposure. – No known cases of transferring to humans • Transmission: direct contact, contact with bodily fluids, possibly airborne routes

Background Detection of DAV • Isolation of the Virus – Enzyme-linked Immuno Assay (ELISA) based test is needed – Exam serum to see if the animal is exposed Generate DAV spike protein • Coat the plate with the spike protein to make ELISA plate that can capture the Ig. G specific to DAV • Making monoclonal antibody to detect the virus in the tissue

Goals of the Study Expressing the DAV spike protein in a PET 6 XHN-N Vector Being able to produce monoclonal antibody

Research Outline • Amplify the coding region of the spike protein by PCR using primer flanking the S protein coding region • Clone the coding region into PCR-TOPO 2. 1 cloning vector • Re-clone the coding region into PET 6 XHN-N vector • Induce the spike protein expression in BL-21 cells

PCR with DAV specific primers Promoter Open reading frame (ORF) of spike protein DAV-SREV DAV-SFOR Hind III DAVs ORF

Amplification of Spike Protein Coding Region 1 2 3 4 MW 1. 4 kb 1. Liver 2. Spleen 3. Kidney 4. non-infected animal tissue * 1, 2, and 3 were success in detecting the Spike Protein *

PCR-TOPO Cloning: TOPO-DAVs • • Ligate Transform Spread Incubate

Screening for TOPO-DAVs • Select of White Colonies in Petri Dish • Picking the white colony and inoculate into 2 ml LB broth with kanamycin • Grow the Bacteria overnight at 37 degree C in a shaking incubator • Extract the plasmid DNA by mini-prep • Digest the extracted DNA with Eco. R I • Run the digested DNA in agarose gel • See if the insert is expressed in the gel run

Result of the Screening by Eco. R I digestion 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 MW * Positive Insert (TOPO-DAVs): 3, 10, 13 *

Re-clone the coding region into p. ET 6 XHN-N vector • Digest the TOPO-DAVs and p. ET 6 XHN-N with Hind III • Run them on the gel • Cut the DNA fragment • Ligate the DAV spike sequence with p. ET 6 XHN-N at Hind III site • Transform the ligation into DH 5 a E. Coli

Digest the DAV-S plasmid and p. ET 6 XHN-N with Hind III Vector Insert

Screening for p. ET 6 XHN-N-DAVs • Pick 20 colonies • Grow the bacteria in LB with ampicillin overnight • Isolate the plasmid by mini-prep • Digest the extract DNA with Hind III • Run the digestion in agarose gel

Summary • DAV-spike gene was amplified by PCR using primers flanking the coding sequence • The PCR product was successfully cloned into TOPO vector • Re-cloning the DAV-spike gene into the expression vector result is pending • After successfully ligating into the expression vector, Purify the vector so that His-spike is left. • Protein synthesis and monoclonal antibodies.